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Urochordates are the closest invertebrate relative to humans and commonly referred to as tunicates, a name ascribed to their leathery outer “tunic”. The tunic is the outer covering of the organism which functions as the exoskeleton and is rich in carbohydrates and proteins. Invasive or fouling tunicates pose a great threat to the indigenous marine ecosystem and governments spend several hundred thousand dollars for tunicate management, considering the huge adverse economic impact it has on the shipping and fishing industries. In this work, the environmentally destructive colonizing tunicate species of Polyclinum constellatum was successfully identified in the coast of Abu Dhabi and methods of sustainably using it as wound-dressing materials, decellularized extra-cellular matrix (dECM) scaffolds for tissue engineering applications and bioinks for bioprinting of tissue constructs for regenerative medicine are proposed. The intricate three-dimensional nanofibrous cellulosic networks in the tunic remain intact even after the multi-step process of decellularization and lyophilization. The lyophilized dECM tunics possess excellent biocompatibility and remarkable tensile modulus of 3.85 ± 0.93 MPa compared to ∼0.1–1 MPa of other hydrogel systems. This work demonstrates the use of lyophilized tunics as wound-dressing materials, having outperformed the commercial dressing materials with a capacity of absorbing 20 times its weight in the dry state. This work also demonstrates the biocompatibility of dECM scaffold and dECM-derived bioink (3D bioprinting with Mouse Embryonic Fibroblasts (MEFs)). Both dECM scaffolds and bioprinted dECM-based tissue constructs show enhanced metabolic activity and cell proliferation over time. Sustainable utilization of dECM-based biomaterials from ecologically-destructive fouling tunicates proposed in this work helps preserve the marine ecosystem, shipping and fishing industries worldwide, and mitigate the huge cost spent for tunicate management.

The present study analyzed the immunomodulatory potential of a newly identified duck beta-defensin, Apl_AvBD2. Recombinant Apl_AvBD2 expressed in HEK293T cells induced a concentration dependent in vitro migration of duck splenocytes, and spleen B- and T-lymphocytes, which was specifically inhibited by anti-Apl_AvBD2 polyclonal antibodies. Among the transcripts of 13 immunologically important genes analyzed in cultured splenocytes for the early immunomodulatory effect of Apl_AvBD2, dendritic cell immunoreceptor (DCIR) mRNA was found to be significantly down-regulated. However, there were no major changes in the expression levels of transcripts for cell surface proteins (MHC I, MHC II 2 beta-chain, TCR-beta, TLR-7, DCAR, CD44, and CD58) and cytokines (IL-2, IFN-gamma, RANTES, MIP-1beta-like and MCP-1 like chemokines). Our results reveal chemotactic and immunomodulatory properties of Apl_AvBD2, two important functions that would help in employing this protein as a molecular adjuvant with avian vaccines.

The cationic, cysteine-rich peptides called beta-defensins play a major role in the innate immune response. Here, we describe the identification and characterization of the duck beta-defensin-2 homologue, Anas platyrhynchos avian beta-defensin 2 (Apl_AvBD2). The 195 base pair open reading frame (ORF) of Apl_AvBD2 has 83% identity with Gga_AvBD2 (chicken) and 85% identity with Mga_AvBD2 (turkey) at nucleotide level. The gene corresponding to the coding region is comprised of three exons and two introns in both Apl_AvBD2 and Gga_AvBD2. The predicted secondary structure of Apl_AvBD2 has the classical "beta-defensin core motif" formed by the beta-sheet rich structure. Apart from mild expression in tissues like kidney, lung, brain, bursa of Fabricious and ovary, Apl_AvBD2 mRNA show a very high level constitutive expression in bone marrow and spleen, indicating that it is a myeloid defensin. Purified recombinant Apl_AvBD2 demonstrated in vitro antibacterial activity against both Gram-positive and Gram-negative bacteria, with a minimum bactericidal concentration (MBC) of 3.7 microM against Micrococcus luteus NCIM 2871 and Escherichia coli NCIM 2685, and of 2.2 microM against Reimerella anatipestifer. The immunomodulatory potential of Apl_AvBD2 was shown by chemotaxis of DT-40 chicken B-lymphocytes. The widespread tissue distribution and the potent bactericidal and chemotactic activity make Apl_AvBD2 an important molecule in the innate immune response in ducks. It may play a vital role in the immune response of these birds against bacterial and viral pathogens.

RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) is a key pro-inflammatory cytokine that belongs to the CC-group of chemokines. The present study was carried out to functionally characterize the previously identified RANTES homologue in domestic duck (GenBank Accession No. AY641435). Recombinant duck RANTES was expressed in Escherichia coli-based and HEK293T cell-based systems. A tRNA supplementation strategy was required to express the protein in E. coli due to the presence of rare codons. In biological assays using HEK293T cell-expressed protein, RANTES was found to mediate chemotaxis of DT-40 chicken B cells and primary duck splenocytes at a concentration of 0.505μg/ml (0.6μM). Immunostaining of the migrated splenocytes using anti-duck CD4 and CD8 monoclonal antibodies and subsequent flow cytometric analysis showed enhanced chemotaxis of CD8+ cells. The recombinant RANTES exhibited in vitro antiviral activity by inhibiting infection of chicken embryo fibroblast cells with duck enteritis virus (DEV) at the same concentration. The effect could be neutralized by rabbit anti-duck RANTES polyclonal serum. The mechanism seems to be direct on viral particles as evidenced by the need for co-incubation of RANTES with DEV prior to the infection for antiviral activity, and also by the enhanced binding of DEV to E. coli expressed purified RANTES on ELISA-based assays. Our results show that the duck RANTES has overlapping biological properties with its mammalian orthologue, and also has possible functional cross-reactivity with chicken immune cells indicated by the chemotaxis of DT-40 cells.

Background Defensins comprise a group of antimicrobial peptides, widely recognized as important elements of the innate immune system in both animals and plants. Cationicity, rather than the secondary structure, is believed to be the major factor defining the antimicrobial activity of defensins. To test this hypothesis and to improve the activity of the newly identified avian β-defensin Apl_AvBD2 by enhancing the cationicity, we performed in silico site directed mutagenesis, keeping the predicted secondary structure intact. Molecular dynamics (MD) simulation studies were done to predict the activity. Mutant proteins were made by in vitro site directed mutagenesis and recombinant protein expression, and tested for antimicrobial activity to confirm the results obtained in MD simulation analysis. Results MD simulation revealed subtle, but critical, structural variations between the wild type Apl_AvBD2 and the more cationic in silico mutants, which were not detected in the initial structural prediction by homology modelling. The C-terminal cationic 'claw' region, important in antimicrobial activity, which was intact in the wild type, showed changes in shape and orientation in all the mutant peptides. Mutant peptides also showed increased solvent accessible surface area and more number of hydrogen bonds with the surrounding water molecules. In functional studies, the Escherichia coli expressed, purified recombinant mutant proteins showed total loss of antimicrobial activity compared to the wild type protein. Conclusion The study revealed that cationicity alone is not the determining factor in the microbicidal activity of antimicrobial peptides. Factors affecting the molecular dynamics such as hydrophobicity, electrostatic interactions and the potential for oligomerization may also play fundamental roles. It points to the usefulness of MD simulation studies in successful engineering of antimicrobial peptides for improved activity and other desirable functions.